PROJECT SUMMARY Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes a debilitating musculoskeletal inflammatory disease. The capacity of CHIKV to achieve extremely high bloodstream titers in humans results in epidemic human-mosquito-human transmission cycles, which promote outbreaks that affect millions of people. Since the reemergence of CHIKV in 2004 in Kenya, the virus has expanded into new areas, including Europe and the Western Hemisphere. In 2013, CHIKV infection was first reported in the island of St. Martin in the Caribbean and continued to spread throughout North and South America, including the United States. This 2013 outbreak of CHIKV in the Western Hemisphere has resulted in over 2.5 million cases, illustrating the explosive epidemic potential when CHIKV emerges in nave populations. Unfortunately, no licensed antivirals or vaccines exist to combat CHIKV. Therefore, it is important to understand specific virus-host interactions that facilitate infection, which will inform development of anti-CHIKV therapeutics. Glycosaminoglycans (GAGs) are used as attachment factors to enhance CHIKV binding to target cells. GAGs are surface-expressed, unbranched carbohydrate polymers that are negatively charged, which facilitates binding to positively charged, basic amino acid residues on recognition proteins. This interaction is common among many pathogenic viruses, including dengue virus, equine encephalitis virus, and HIV. CHIKV vaccine strain 181/25, which exhibits a high capacity to bind GAGs, and clinical isolates SL15649 and AF15561, which exhibit moderate capacity for GAG binding, require GAGs for efficient infection in vitro. A single polymorphism in the E2 attachment protein between 181/25 and AF15561 appears to influence GAG utilization and pathogenesis and tropism in acute CHIKV disease in mice, suggesting an influential role of GAG binding during CHIKV infection. In this proposal, I will test the hypothesis that interactions between residues on the CHIKV E2 glycoprotein (attachment protein) and specific GAGs regulate viral tropism and pathogenesis. To identify the GAGs to which CHIKV binds, CHIKV virus-like particles (VLPs) will be engineered for multiple CHIKV strains and tested in glycan microarray screens. Additionally, I will define the amino acid residues in the E2 glycoprotein required for GAG binding by engineering and characterizing a panel of mutant viruses in which basic surface residues in E2 will be exchanged with alanine. These studies will identify the residues on E2 that contribute to GAG binding. To determine the influence of GAG binding on infection and disease, the mutant GAG-binding viruses generated will be used to infect 3-to-4 week-old C57BL/6 mice. Tropism and pathological outcomes elicited by viral infection will be assessed. The proposed research will enhance an understanding of CHIKV attachment to host cells and clarify the function of GAG utilization during CHIKV infection.